Normal human physiology is dependent upon a continual supply of oxygen. When oxygen becomes limiting, cells of affected tissues undergo a number of adaptive responses to aid survival. These include metabolic adjustments, for example, switching energy production from oxidative phosphorylation to glycolysis, as well as genetic reprogramming events aimed at increasing oxygen supply to tissues. Key genes which are activated during hypoxia (low oxygen stress) include those which encode erythropoietin (EPO), a growth factor which increases the production of oxygen carrying red blood cells; Vascular Endothelial Growth Factor (VEGF), a protein which promotes new blood vessel development; and a set of genes which produce enzymes involved in glycolysis as well as a number of other changes.
A number of oxygen sensitive mediators have been identified. Two proteins which sense depleted oxygen levels and subsequently act as transcription factors (gene regulatory factors) to induce the above mentioned genes are the Hypoxia Inducible Factors 1α and 2α (HIF-1α and HIF-2α).
HIF-1α and HIF-2α are two closely related transcription factors. The schematic of FIG. 1 shows these proteins exhibit a similar organisation of functional domains. The N-terminal bHLH/PAS domains are important for dimerisation and DNA binding; the Oxygen Dependent Degradation Domains (ODDs) mediate protein turnover; and the C-terminal transactivation domains (CADs) are important for inducing transcription of target genes.
Both the ODD and CAD domains of each protein are known to sense depleted cellular oxygen levels. The ODDs confer extreme lability to the HIF-1α and HIF-2α proteins. At normoxia (20% atmospheric oxygen), the proteins are so labile that they are effectively absent from most cell types when analysed by antibodies. In contrast, when cells are subjected to hypoxia (<2% oxygen) the HIF proteins are stable and readily detected with antibodies. Recently, the mechanism by which the ODDs sense oxygen was elucidated (Zhu and Bunn, 2001; Ivan et al., 2001; Jaakkola et al., 2001). At normal oxygen levels, an oxygen and Fe2+ dependent prolyl hydroxylase enzyme is responsible for hydroxylating a conserved proline residue within the ODDs. The hydroxyproline is required for connecting HIF-1α and HIF-2α to the von-Hippel Lindau factor (VHL). VHL is part of a protein complex that initiates addition of ubiquitin chains to the HIF substrate proteins, subsequently targeting the HIFs to the protein degrading proteasome. During hypoxia, the oxygen dependent prolyl hydroxylase does not function and the critical proline in the ODDs remains unmodified. This allows the HIF proteins to avoid VHL surveillance and escape the proteasome degradation machinery (FIG. 2).
During hypoxia, HIF-1α and HIF-2α become stable proteins and function as transcription factors, that is, they trigger induction of target genes such as EPO and VEGF. The ability to induce target genes relies on transactivation domains, which are responsible for connecting the DNA bound HIF proteins to the transcription machinery of the cell. Two distinct transactivation domains exist within HIF-1α and HIF-2α. One is ill defined and intrinsic to the ODD, while the other is at the C-terminus (CAD) and represents a second domain which is capable of responding to hypoxia. The CADs of HIF-1α and HIF-2α are the dominant transactivation domains and respond to hypoxia in a manner which is independent of protein stability. The importance of the CAD from HIF-1α is exemplified by disruption of its function in mice. In these experiments, ectopic expression of CAD peptides resulted in attenuation of hypoxia induced genes and a reduction in growth of induced tumours (Kung et al., 2000).
There are several documents that describe some aspects of the behaviour of the CAD domain of HIF in normoxic conditions compared to hypoxic conditions however none of these has identified the hydroxylation of a target asparagine ( O'Rourke et al., 1999, Ema et al., 1999, Carerro et al., 2000, Gu et al., 2001, Jiang et al., 1997). The term CAD refers to a functional transactivation domain at the C-terminus of the HIF-1α and HIF-2α proteins and different laboratories use the term to describe varying lengths of the C-terminus. We define the CAD as the hypoxia inducible C-terminal transactivation domain contained within the last 100 amino acids of HIF-1α and HIF-2α.